The long-range goal of the proposed studies is to determine the functions of long non-coding RNAs in vertebrate development. The past decade has seen an explosion of genome-wide studies that have identified a plethora of previously unannotated transcripts. Among these novel transcripts, the class of long non-coding RNAs (lncRNAs) has attracted particular attention since they represent an extensive, largely unexplored component of the genome: the dark matter of the genome. The proposed study aims to perform a reverse genetic screen and analyze the function of 100 lncRNAs in zebrafish. Genomics and computational approaches have led to the identification of several hundred lncRNAs expressed during zebrafish development. 100 of these lncRNAs, selected by conservation, genomic location, expression, and chromatin profile, will be disrupted. Antisense oligonucleotides will be used to directly interfere with lncRNAs, and site-specific endonucleases (TALENs and CRISPRs) will be employed to disrupt lncRNA genes. Embryos and larvae will then undergo systematic phenotypic characterization to reveal the functions of lncRNAs. The proposed reverse genetic screen will provide the first comprehensive analysis of vertebrate lncRNA function in vivo and generate the resources for the field to reveal the roles of this mysterious class of molecules.